Opiate/narcotic analgesics, typified by morphine sulfate, are the most effective analgesics for treating acute and chronic severe pain, but their clinical utility is often hampered by the development of analgesic tolerance and painful hypersensitivity to both innocuous and noxious stimuli. The mechanisms by which chronic opiate exposure induce hyperalgesia and antinociceptive tolerance are unclear but neuroimmune activation, cellular apoptosis and oxidative/nitrative stress in the spinal cord have been proposed, Ceramide is a sphingolipid signaling molecule with powerful proapoptotic and proinflammatory properties and may also contribute to oxidative/nitrative stress. Ceramide is generated from de novo synthesis coordinated by serine palmitosyltransferase and ceramide synthase and/or by enzymatic hydrolysis of sphingomyelin by sphingomyelinases (SMases). Using a well established murine model, our preliminary experiments revealed that repeated administration of morphine increased the levels of ceramide in the dorsal horn of the lumbar segment of the spinal cord and that its inhibition by fumonisin B1, an inhibitor of ceramide synthase, attenuated the development of antinociceptive tolerance. These events were associated with inhibition of apoptosis and oxidative/nitrative stress in dorsal horn tissues. Furthermore, inhibition of ceramide synthesis by D609 and myriocin, inhibitors of SMAse/sphingomyelin synthase and serine palmitoyltransferase respectively blocked antinociceptive tolerance. Together, these findings support the central thesis of this exploratory proposal: increased formation of ceramide in the spinal cord is an important pathway in the development of morphine-induced hyperalgesia and antinociceptive tolerance. To address this novel hypothesis, we propose a comprehensive experimental strategy employing molecular, bio-analytical, biochemical, pharmacological and genetic approaches. Two Specific Aims will test our hypothesis. In Specific Aim 1, we will demonstrate by pharmacologic and genetic approaches that inhibition of the increased formation of ceramide blocks the development of morphine- induced hyperalgesia and antinociceptive tolerance thus identifying the predominant enzymatic pathway responsible for its biosynthesis. In Specific Aim 2, we will elucidate the molecular and biochemical mechanisms whereby ceramide modulates hyperalgesia and antinociceptive tolerance. Specifically, we will determine ceramide's effects on three biochemical pathways within spinal tissue: (a) neuroimmune activation, (b) oxidative/nitrative stress and (c) apoptosis. Successful validation of our hypothesis will define for the first time the important role of ceramide in morphine-induced hyperalgesia and antinociceptive tolerance providing the scientific foundation towards the development of inhibitors of ceramide biosynthesis as adjunct to opiates for the management of chronic pain, in particular for those patients who require long-term opioid treatment for pain relief. PUBLIC HEALTH RELEVANCE Opioid drugs such as morphine are the most effective analgesics for treating severe chronic pain, but their pain-relieving action is often diminished during chronic administration, necessitating dose escalation that reduces quality of life for the patient. Our studies will determine for the first time that inhibition of ceramide biosynthesis, with novel agents, restores the pain-relieving action of morphine. The broader implications of our findings may open a new frontier in chronic pain management thus alleviating the socioeconomic consequences it causes.